PROJECT SUMMARY/ABSTRACT A variety of important biological pathways in health and disease are regulated by intramembrane proteolysis, the process that achieves the targeted cleavage of protein substrates within or proximal to the lipid bilayer. Four classes of intramembrane proteases have been described, including the site 2 protease (S2P) metalloprotease family, the rhomboid serine protease family, the Rce1 glutamyl protease family, and the aspartyl protease family that includes presenilins and the related Signal Peptide Peptidase (SPP)-like (SPPL) subfamily. The aspartyl proteases of the SPPL subfamily, including SPP, SPPL2a, SPPL2b, SPPL2c, and SPPL3, are perhaps the least understood subset of intramembrane proteases. Only recently have the biological roles of some of these proteases emerged, and only a handful of relevant biological substrates have been identified. How these intramembrane proteases specifically recognize and cleave their substrates remains mysterious. Recently, we discovered a critical role for SPPL3 in NK cell maturation and cytotoxicity. SPPL3 is required in a cell-autonomous manner for the maturation of NK cells from the immature CD27+CD11b- stage to the CD27+CD11b+ and CD27-CD22b+ stages, and for normal NK cell cytotoxicity toward tumor cell targets. Mice engineered to express only SPPL3 D271A in the NK lineage revealed that the proteolytic function of SPPL3 is required in NK cell maturation and function. Like other aspartyl intramembrane proteases of the SPPL subfamily, very little is known about how SPPL3 recognizes and cleaves its substrates. All SPPL proteases possess YD and GXGD motifs that contain the catalytic aspartates, but other regions in the protein that are required for substrate recognition and cleavage have not been identified. It is also unclear whether a specific amino acid sequence in a putative substrate is recognized by SPPL3 or whether other features of the target, such as the size or composition of cytoplasmic and luminal domains, facilitate recognition by SPPL3. Furthermore, although some SPPL3 substrates have been identified, the relevant substrate that must be cleaved by SPPL3 during NK cell maturation is currently unknown. To expand our basic understanding of SPPL3 and related aspartyl intramembrane proteases, and to advance understanding of the key checkpoint in NK cell maturation controlled by SPPL3, we will 1) use a novel high-throughput assay for SPPL3 protease activity to identify residues required for substrate recognition and cleavage; 2) determine whether substrate binding and cleavage are controlled by different SPPL3 residues, and whether the same SPPL3 determinants are required for the cleavage of four biochemically distinct substrates; 3) address what features in a substrate allow it to be recognized and cleaved by SPPL3; and 4) identify SPPL3 substrates in NK cells. Our results will expand understanding of intramembrane aspartyl proteases and illuminate molecular events that control NK cell development and function.